Plasmodium sporozoites make a remarkable journey from the mosquito midgut wall to the mammalian liver. It is the overall goal of this proposal to elucidate the role of the sporozoite's major surface protein, the circumsporozoite protein (CSP) in this journey. We have been studying the proteolytic processing of CSP and thus far, have found that processing occurs when sporozoites contact hepatocytes and is required for efficient invasion. Specifically, our data indicate that the amino-terminus of CSP masks the cell-adhesive domain in the carboxy-terminus and that proteolytic cleavage results in removal of the amino-terminus and exposure of this cell-adhesive domain. Overall our data suggest that CSP has two functional domains, each with distinct role(s) during the sporozoite's journey. The goal of this proposal is to continue our structure-function work on this important protein to define the role of each CSP domain in the mammalian host and to identify the protease responsible for CSP cleavage. The specific aims are: 1) To elucidate the phenotype of sporozoite mutants which expressed only the cleaved form of CSP in the dermis of the mammalian host. Using quantitative PCR, transcriptional profiling and confocal microscopy, we will determine whether N-terminal deletion mutants remain in the skin and begin their development there. 2) To determine the binding properties of each CSP domain by generating mutants in N-terminal residues that we have identified as having potential heparin-binding activity and using biochemical techniques to determine the binding partner of the TSR domain. 3) To identify the protease that cleaves CSP and determines its subcellular location in the sporozoite. These studies will be performed using conditional mutagenesis to delete candidate protease genes and using antisera to localize the proteases in sporozoites. Overall these studies will elucidate how CSP functions in the sporozoite's journey from mosquito to mammalian host and should open up new avenues for the control and prevention of malaria. PUBLIC HEALTH RELEVANCE: These studies are of relevance to the control and prevention of one of the most important infectious diseases in the world. Malaria continues to impact significantly, children and adults living in tropical and subtropical parts of the world, in part due to drug resistance to our best anti-malarials and to the lack of an effective vaccine. The experiments described in this proposal could provide the basis for new drug therapies and improve upon existing approaches to malaria vaccine development.